EXCEED THE SPACE PROVIDED. There are many strains of murine coronavirus (MHV) having different tropisms and levels of virulence. The neurotropic strains, such as JHM and A59, cause acute encephalitis followed by chronic demyelinating disease; infection of mice with these MHV strains is one of the best animal models to study the humans demyelinating disease, multiple sclerosis. JHM is highly neurovirulent, while A59 is more neuroattenuated. In this proposal we are interested in unraveling the factors leading to high neurovirulence, that is the ability of the virus to cause high levels of encephalitis and mortality during acute infection. We have characterized neurovirulence of isogenic recombinant viruses differing only in the spike gene, encoding the spike for the highly neurovirulent JHM or the mildly neurovirulent A59 virus, with all background genes derived from A59. It is clear that the high neurovirulence of the JHM spike encoding virus (SJHM-RA59) is associated with more extensive viral spread in the CNS, a more intense immune response (with an immunopathological component), as compared to parental wild type recombinant A59 (RA59). We have formulated the hypothesis that the level of neurovirulence following MHV infection of mice is highly dependent on the degree of spread of viral antigen in the central nervous system. The degree of spread is influenced by ; 1) the interaction of the virus with the receptor during entry; 2) inherent ability to spread in neural cell types and 3) the magnitude and type of immune response. We will use RA59 and SJHM-RA59 as well as another set of isogenic recombinants differing in the spike but with background genes derived from JHM (RJHM and SA59-RJHM) to test this hypothesis. Thus we will carry out studies comparing these recombinant viruses as to 1) virus spread in vivo and in vitro in primary neuronal cultures 2) infection of mice with a partial knockout of the ceacaml receptor gene and 3) the extent and type of immune response elicited. We will determine the contributions of spike gene vs. other background genes in determining viral spread and the intensity and type of immune response that ultimately lead to neurovirulence.